Introduction

There has been serious concern about the deterioration of human and animal male reproductive health in association with exposure to environmental, industrial, and occupational chemicals, as well as several pharmaceuticals [1,2,3]. Among an array of toxic chemicals, heavy metals are environmental toxicants which pose male reproductive toxicity and infertility in wildlife, experimental animals, and humans [1, 2]. Lead (Pb) is a toxic pollutant extensively distributed in the environmental and biological systems. As a result of industrial development, the environmental levels of Pb have raised more than 1000 times, over the past 30 years [4]. It has been reported that Pb exposure, even at very low doses, is implicated in the impairment of a wide range of body function in laboratory animals and humans [5, 6]. Several systems including the heart, kidneys, liver, central and peripheral nervous systems, endocrine system, reproductive system, and hematological attributes are affected by Pb exposure [7,8,9,10,11,12,13,14].

Previous investigations have shown that Pb exposure induces deleterious effects on sperm motility, caused premature acrosome reaction, reduced zona-intact oocyte penetrating capability, decreased plasma testosterone, and impaired spermatogenesis [15,16,17,18,19]. The role of oxidative stress and its associated complications have been well described in Pb-induced reproductive toxicity [15,16,17,18,19]. A defect in testicular enzymatic antioxidants, such as superoxide dismutase, as well as an increase in sperm reactive oxygen species (ROS) generation, has been documented in Pb-exposed animals [15,16,17,18,19]. Several reproductive dysfunctions including histological evidence of testicular damage inhibited spermatogenesis, and a reduction in sperm quality and quantity have also been shown in animals exposed to Pb [15,16,17,18,19]. Hence, antioxidants may have the potential as protective agents against Pb-induced reproductive toxicity. Protective effects of several chemicals such as vitamin C, vitamin E, β-carotene, zinc, selenium, and polyphenol compounds have been investigated against Pb-induced reproductive toxicity [6, 17,18,19,20]. Sperm mitochondria also seem to be a potential target of Pb toxicity [21,22,23]. As proper mitochondrial function and enough level of energy guarantee proper sperm motility and function, mitochondrial injury leads to reduced sperm parameters and reproductive failure [24, 25]. Hence, in the current study, the involvement of sperm mitochondrial function in Pb-exposed rats was also evaluated as potential mechanisms of protection provided by carnosine and histidine.

Carnosine (CAR), a dipeptide composed of L-histidine (HIS) and β-alanine, is a potential endogenous antioxidant found mainly in skeletal and cardiac muscle, brain, and other organs in humans [26,27,28]. Numerous vital functions are reported for CAR [29,30,31,32]. Due to antioxidant and membrane-stabilizing activity of CAR under physiological conditions, it has been established that CAR prevents lipid peroxidation and oxidative damage to proteins [33]. This dipeptide also inactivates highly reactive aldehydes produced from degenerated biomembrane lipids [34]. Furthermore, the beneficial effect of CAR in the reproductive system has been widely investigated [35, 36]. Hence, in the current study, we aimed to assess the potential protective effect of CAR in Pb-induced reproductive toxicity and its potential mechanism(s) of protective properties.

It has been shown that some amino acids protect several types of animal cells against hypothermia and have ameliorative effects on sperm parameters in cryopreservative extenders [37]. There is a good body of literature explaining the protective function of amino acids in cryoprotection [38,39,40,41,42,43]. Among them, it has been reported that HIS cause a decrease in polyunsaturated fatty acid dilapidation of spermatozoa under peroxidation stress in humans [44]. The protective effects of this amino acid in other experimental models have also been evaluated [45,46,47]. Hence, another aim of the current study was to determine the protective effect of HIS against Pb-induced reproductive toxicity.

Materials and Methods

Chemicals

2′,7′ Dichlorofluorescein diacetate (DCFH-DA), 3-(N-morpholino) propane sulfonic acid (MOPS), 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 4,2-Hydroxyethyl,1-piperazineethanesulfonic acid (HEPES), bovine serum albumin (BSA), lead (Pb) acetate, dimethyl sulfoxide (DMSO), thiobarbituric acid (TBA), dithiobis-2nitrobenzoic acid (DTNB), glutathione (GSH), malondialdehyde (MDA), eosine, Coomassie brilliant blue, negrosine, ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), sucrose, KCl, NaCl, dithiothreitol (DTT), Na2HPO4, MgCl2, Rhodamine 123 (Rh 123), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Trichloroacetic acid (TCA) and hydroxymethyl aminomethane hydrochloride (Tris–HCl) were purchased from Merck (Darmstadt, Germany). The salts for preparing buffer solutions were of the analytical grade and obtained from Merck (Darmstadt, Germany).

Animals and Treatments

Mature male Sprague-Dawley (SD) rats (n = 48; 300–320 g) were purchased from Experimental and Comparative Medicine Research Center of Shiraz University of Medicine, Shiraz, Iran. Animals were maintained under controlled conditions (12:12 h, photoschedule; 18–22 °C; appropriate ventilation, ≈ 40% humidity). Animals had free access to tap water and commercial rodent pellets (Behparvar®, Tehran, Iran). All animal procedures were performed in compliance with the regulations and guidelines of the local ethics committee at Shiraz University of Medical Sciences, Shiraz, Iran (95-01-36-11290). Animals were allotted to six groups (n = 8 in each group). The treatments were as follows: (a) control (vehicle-treated), (b) Pb-acetate (20 mg/kg/day, gavage), (c) Pb-acetate + carnosine (250 mg/kg/day, gavage), (d) Pb-acetate + carnosine (500 mg/kg/day, gavage), (e) Pb-acetate + histidine (250 mg/kg/day, gavage), (f) Pb-acetate + histidine (500 mg/kg/day, gavage). Pb-acetate, carnosine, and histidine were administered daily for 14 consecutive days. On day 15, the animals were anesthetized, and samples collected. HIS and CAR were administered at least 6 h after Pb-acetate.

Sample Collection

Animals were anesthetized (Thiopental 70 mg/kg, i.p.) and the epididymides and testes were excised and weighed. The left testes were kept in 10% formalin for histopathological evaluations. Total antioxidant capacity, lipid peroxidation, reactive oxygen species (ROS) production, protein carbonylation, and glutathione contents were determined in the right testes. Sperm was collected from the left cauda epididymis.

Testis Weight Index

Testicular weight index (WI) was determined as WI = [wet weight of organ (g) / body weight (g)] × 100.

Sperm Quality Evaluation

Epididymal sperm count and sperm progressive motility were determined as previously reported [48]. Briefly, epididymal sperm was extracted by mincing the caudal epididymis in warm phosphate-buffered saline (PBS; 35 °C; pH = 7.4). Sperm progressive motility was recorded by putting a drop of sperm suspension on a glass slide covered with a coverslip and observing the sperm under a Zeiss (Jena, Germany) compound light microscope (400 × magnification) equipped with hot-stage (35 °C). The hypo-osmotic swelling (HOS) test was performed by mixing 10 μL of sperm suspension with 50 μL of hypo-osmotic solution (50-mOsm NaCl) for 10 min at 37 °C by evaluating the sperm percentage with a swollen “bubble” around the curled flagellum by counting 200 cells on each slide, using light microscopy (× 1000 magnification) [49, 50]. Sperm concentration was determined by placing 10 μL of diluted epididymal fluid on a Neubauer hemocytometer using a light microscope (× 200 magnification). Sperm abnormality and viability were monitored in duplicate (200 sperm per sample) after eosin-nigrosin staining [51]. Spermatozoa with protoplasmic droplets, ab-axial tail, malformed heads, double tails, coiled tails, bent tails, without tail and head, were recorded as abnormal under a phase-contrast microscope (Olympus BX41; Olympus Optical Co. Ltd., Japan) [52, 53].

Sperm Mitochondrial Dehydrogenase Activity (MTT Assay)

The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used for colorimetric measurement of mitochondrial dehydrogenase activity in sperm [52, 53]. For this purpose, sperm samples (1 mg protein/mL) were incubated with 40 μL MTT solution (5 mg/mL) at 37 °C for 30 min. The reaction products (purple formazan crystals) were dissolved in dimethyl sulfoxide (DMSO; 1 mL), and the optical density (OD) of the dissolved formazan was determined at λ = 570 nm using an EPOCH plate reader (BioTek Instruments, Highland Park, USA) [54, 55].

Sperm Mitochondrial Membrane Potential

Mitochondrial uptake of the cationic fluorescent dye, rhodamine-123, was applied for the determination of sperm mitochondrial membrane potential [55, 56]. Briefly, sperm samples (1 mg protein/mL) were incubated with 10 μM of rhodamine-123 for 30 min at 35 °C under dark conditions. After centrifugation (10,000×g, 4 °C), the fluorescence intensity of supernatant was measured (λ excitation = 485 nm and λ emission = 525 nm) using a FLUOstar Omega® multifunctional microplate reader (BMG LABTECH, Germany) [55, 56].

Testicular and Sperm Reactive Oxygen Species

Testicular and sperm ROS levels were measured using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA) [56, 57]. Briefly, DCFH-DA was added (10 μM final concentration) to sperm and homogenized testicular samples (1 mg protein/ml). Samples were incubated for 30 min at 35 °C in the dark. Afterward, the DCF fluorescence intensity was recorded using a FLUOstar Omega® multifunctional microplate reader (BMG LABTECH, Germany) (λ excitation = 485 nm and λ emission = 525 nm) [52, 58].

Testicular and Sperm Lipid Peroxidation

Thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation, were measured in the testis. For this purpose, 500 mg of testis tissue homogenate (10% w/v in KCl, 1.15% w/v) or samples of 106 sperm/ml was added to a reaction mixture consisting of 1 mL thiobarbituric acid (0.375%, w/v) and 3 mL phosphoric acid (1% w/v, pH = 2) [53, 59]. The mixture was incubated at 100 °C for 45 min. Afterward, 2 mL of n-butanol was added and vigorously mixed. Finally, specimens were centrifuged (10,000×g for 5 min) and absorbance of the developed color in n-butanol phase was measured at λ = 532 nm using an Ultrospec 2000®UV spectrophotometer (Scinteck Instruments, USA) [60].

Testicular and Sperm Glutathione Content

Testicular and sperm glutathione (GSH) levels were determined spectrophotometrically using 5, 50-dithiobis-2-nitrobenzoic acid (DTNB) as the indicator [53, 61]. Briefly, testis tissue (200 mg) or sperm (106 cells/ml) samples were homogenized (Heidolph, Germany) in 8 mL of ice-cooled EDTA solution (20 mM). Then, 5 mL of the homogenate was mixed with 4 mL distilled water and 1 mL trichloroacetic acid (50% w/v). The mixture was gently shaken and centrifuged (10,000×g, 10 min, 4 °C). Then, 2 mL of the supernatant was mixed with 4 mL Tris buffer (pH = 8.9) and 100 μL Ellman’s reagent (DTNB, 10 mM in methanol) [62, 63]. The intensity of the developed yellow color was measured at λ = 412 nm using an Ultrospec 2000®UV spectrophotometer Pharmacia Biotech® UV spectrophotometer (Scinteck Instruments, United States).

Ferric-Reducing Antioxidant Power

The ferric-reducing antioxidant power (FRAP) of testicular tissue and sperm samples was measured in each experimental group. The FRAP assay evaluates changes in the absorbance at λ = 593 nm due to the formation of a blue-colored Fe2+-tripyridyltriazine from the colorless oxidized Fe3+ form by the action of electron-donating antioxidants [53, 64]. Briefly, the working FRAP mixture was freshly prepared by mixing 10 parts of 300 mmol/L acetate buffer (pH = 3.6) with 1 part of 10 mmol/L TPTZ (2, 4, 6-tripyridyl-s-triazine) in 40 mmol/L hydrochloric acid and with 1 part of 20 mmol/L ferric chloride. Testicular or sperm samples was homogenized in Tris–HCl buffer (0.25 M; pH = 7.4; 4 °C), containing 0.2 M sucrose and 5 mM dithiothreitol (DTT) [65]. Afterward, 1.5 mL FRAP reagent and 150 μL deionized water were added to 50 μL testicular tissue homogenate and incubated at 37 °C for 5 min. The intensity of the resultant blue color was measured at λ = 593 nm using an Ultrospec2000® spectrophotometer (Scinteck Instruments, United States) [66]. The protein content of the sample was measured, using the Bradford method, for standardization of data [67].

Testicular Histopathology

For histopathological evaluations, testicular specimens were fixed in buffered formalin solution (0.4% NaH2PO4, 0.64% Na2HPO4, and 10% formaldehyde in distilled water; pH = 7.4). The samples were rinsed with running water, dehydrated in graded alcohol, cleared in xylene, and embedded in paraffin; paraffin-embedded samples were cut into 5-μm sections with a rotary microtome and stained with hematoxylin-eosin stain. Tissue histopathological alterations were evaluated by a pathologist in a blind manner using a light microscope (Olympus BX41; Olympus Optical Co. Ltd., Japan). Testicular tubular injuries were monitored, and a four-level grading scale was used to quantify the histopathological alteration [68, 69]. Grade 1 injury shows a typical testicular structure with an orderly arrangement of germinal cells. Grade 2 injury demonstrates less orderly, no cohesive germinal cells, and firmly packed seminiferous tubules. Grade 3 injury exhibits disordered, sloughed-off germinal cells with shrunken, pyknotic nuclei, and less distinct seminiferous tubule borders. Grade 4 injury shows seminiferous tubules that are tightly packed with coagulative necrosis of the germinal cells [68, 69]. For testis tubular desquamation, the seminiferous tubules were evaluated for the existence of complete spermatogenesis and focal or diffuse atrophy or tubular degeneration, depending on the number of affected tubules. Early intratubular desquamation was graded as mild (+), moderate (++), or severe (+++) [68, 69]. The existence of multinucleated cells, within the tubular lumen or among spermatogenic cells, was recorded [68, 69]. The existence of degenerative alterations in interstitial Leydig cells was also studied (e.g., nuclear atrophy or cytoplasmic vacuolization) [68, 69]. The spermatogenic index was reported based on the ratio of the number of seminiferous tubules with spermatozoids to the empty tubules [68, 69].

Statistical Analysis

Data were expressed as mean ± SD. Data analysis was performed by the one-way analysis of variance (ANOVA), and mean comparison performed using the Tukey’s multiple comparison test as the post hoc test at P < 0.05 (SPSS software, version 19, IBM Corporation, NY, USA).

Results

Body and testicular weight were lower in Pb-treated rats (Fig. 1), but carnosine (CAR; 250 and 500 mg/kg) and histidine (HIS; 500 mg/kg) supplementation mitigated the lead-induced decrease in testis and body weights (Fig. 1). The effect of CAR and HIS on animal body weight or testicular weight index was not dose-dependent in the current study (Fig. 1).

Fig. 1
figure 1

Effect of lead on live and testicular weights in the rat. Pb lead; CAR carnosine; HIS histidine. Data are given as mean ± SD (n = 8). ***Significantly different from control (P < 0.001). aSignificantly different from Pb-treated group (P < 0.001). ns not significant as compared with Pb-treated group (P > 0.05)

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Assessment of sperm parameters in Pb-treated rats revealed that exposure to Pb caused a significant decrease in sperm motility, viability, and membrane integrity (Fig. 2). A significant increase in the amount of abnormal sperm was also evident in Pb-treated animals (Fig. 2). On the other hand, improvements in these attributes were observed consequent to CAR (250 and 500 mg/kg) and HIS (250 and 500 mg/kg) administration (Fig. 2).

Fig. 2
figure 2

Effect of lead on epididymal sperm parameters in the rat. Pb lead; CAR carnosine; HIS histidine. Data are presented as mean ± SD (n = 8). ***Significantly different from control (P < 0.001). aSignificantly different from Pb-treated group (P < 0.001)

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Evaluating sperm markers of oxidative stress revealed significant ROS formation, lipid peroxidation, glutathione depletion, and decreased antioxidant capacity in Pb-treated rats (Fig. 3). It was found that (250 and 500 mg/kg) and HIS (250 and 500 mg/kg) supplementation significantly mitigated the oxidative stress markers in sperm isolated from Pb-treated animals (Fig. 3).

Fig. 3
figure 3

Reactive oxygen species (ROS) formation, lipid peroxidation, glutathione content, mitochondrial dehydrogenases activity, and mitochondrial depolarization in the sperm of lead-treated rats. Pb lead; CAR carnosine; HIS histidine. Data are shown as mean ± SD (n = 8). ***Significantly different from control (P < 0.001). aSignificantly different from Pb-treated group (P < 0.001). bSignificantly different from Pb-treated group (P < 0.05)

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Impairment in sperm mitochondrial function was revealed by an increment in mitochondrial depolarization and a decline in mitochondrial dehydrogenase activity in the Pb-treated group (Fig. 3). It was found that (250 and 500 mg/kg) and HIS (250 and 500 mg/kg) abated the collapse of sperm mitochondrial membrane potential and preserved mitochondrial dehydrogenase activity in the sperm of Pb-treated rats (Fig. 3).

Testis tissue histopathological changes revealed the severe tubular injury, tubular desquamation, and low spermatogenesis index in Pb-treated rats (Fig. 4 and Table 1). CAR and HIS supplementation decreased lead-induced testicular changes (Fig. 4 and Table 1).

Fig. 4
figure 4

Photomicrographs of the testicular architecture of lead-treated rats. a Tissue from control (vehicle-treated) animals revealed normal testis tissue architecture (Table 1). b Histopathological changes in the lead (Pb)-exposed rats (20 mg/kg/day for 14 consecutive days) revealed as tubular injury, tubular desquamation, and low spermatogenic index (Table 1). c, d Carnosine (250 and 500 mg/kg respectively) treatment alleviated lead (Pb)-induced testis tissue histopathological changes (Table 1). e, f Histidine (250 and 500 mg/kg respectively)-treated rats showed mild testicular injury (Table 1).

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Table 1 Histopathological changes of the testis tissue in the lead (Pb)-treated rats
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A significant increase in biomarkers of oxidative stress in the testis tissue of Pb-treated animals was evident as elevated ROS formation and lipid peroxidation (Table 2). On the other hand, testis tissue antioxidant capacity was decreased, and glutathione reservoirs were depleted in Pb-treated animals in comparison with the control group (Table 2). It was found that CAR (250 and 500 mg/kg) and HIS (500 mg/kg) mitigated oxidative stress and its consequences in the testis tissue of Pb-treated rats (Table 2). The effect of CAR and HIS on tissue biomarkers of oxidative stress was not dose-dependent in the current study (Table 2).

Table 2 Biomarkers of oxidative stress in the testis of lead (Pb)-treated rats
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Its noteworthy to mention that administration of CAR or HIS (500 mg/kg/day) alone did not significantly affect the testis and sperm parameters of interest in the current investigation (Data not shown). It should also mention that the protective properties of HIS and CAR against Pb-induced decrease in rat sperm functionality were not dose-dependent in the current study (Figs. 2 and 3). On the other hand, no significant differences between the protective properties of HIS and CAR were found neither at sperm nor the mitochondria level (Figs. 2 and 3).

Discussion

Pb exposure is well established to have detrimental effects on several organs including the reproductive system. It has been shown that Pb interrupts reproductive hormonal balance and prompts oxidative stress in the rat testes [70]. Hence, antioxidants might serve as potential protective agents against lead-induced reproductive toxicity. Results of the current investigation supported the protective effects of carnosine and histidine against Pb reproductive toxicity.

We found an increase in the testicular and sperm mitochondrial ROS biosynthesis and sperm MDA levels, in addition to severe testis morphological alterations in Pb-treated rats. Moreover, poor sperm parameters including sperm motility, viability, abnormality, and membrane integrity and impairment in reproductive mitochondrial indices such as mitochondrial depolarization and decrease in mitochondrial dehydrogenase activity were also found in the current study. On the other hand, biomarkers of oxidative stress were increased where total antioxidant capacity was decreased in Pb-treated rats.

The effect of Pb on body weight and relative testicular weight percentage were significantly lower than in other groups. These observations are in agreement with the report of Acharya et al. [71] who reported a significant decrease in the body and testicular weights in animals treated with a single dosage of Pb, which they attributed to excessive ROS generation. However, there are reports of an increase in testicular weight due to acute Pb-administration which were attributed to interstitial edema due to fluid accumulation in the testis [70, 72].

The high content of polyunsaturated fatty acids in the sperm plasma membrane makes it susceptible to lipid peroxidation and triggering a higher TBARS synthesis by ROS generation [73]. The defect in testis and sperm antioxidant enzymes and ROS-induced biomembrane disruption (Fig. 3) might be a reason for alteration in sperm membrane integrity in Pb-treated animals [74]. Our results indicated that CAR and HIS supplementation could inhibit Pb-induced oxidative stress and reproductive toxicity (Fig. 5).

Fig. 5
figure 5

Schematic representation of the effect of carnosine and histidine against lead-induced reproductive toxicity

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Glutathione (GSH) and its associated enzyme systems are the most important cellular defense mechanism against xenobiotics; GSH is also an essential antioxidant for quenching free radicals [75]. The deleterious effects of Pb on cellular glutathione stores were also documented by previous investigators [21, 76, 77]. Lead might inactivate glutathione by binding to the sulfhydryl groups. In the current investigation, we found that Pb significantly depleted testicular and sperm glutathione contents. In line with our observation, Dkhil et al. reported considerable reduction in testicular superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase activities, following administration of Pb [70]. Similar to our findings regarding the FRAP test data as an index of total antioxidant capacity, Dkhil et al. [70] observed that Pb intoxication induced a falling down of the antioxidative defense in the rat testes. Therefore, it seems the antioxidative properties of CAR and HIS might play a primary role in their protective actions against Pb reproductive toxicity [70].

It is known that Pb adversely affects mitochondrial function. The inhibition of mitochondrial oxidative phosphorylation, collapse of mitochondrial membrane potential, inhibition of mitochondrial respiratory enzyme activities, ATP depletion, and energy crisis are adverse effects reported in different models of Pb-induced toxicity [21,22,23, 78, 79]. Our data also support these findings; hence, mitochondrial dysfunction might serve as an essential mechanism in Pb reproductive toxicity. On the other hand, the regulation of mitochondrial function is an essential feature of CAR and HIS [60, 80,81,82,83,84]. In this study, CAR and HIS supplementation preserved sperm mitochondrial membrane potential and dehydrogenase activity. Therefore, these chemicals might prevent Pb-induced toxicity through regulating mitochondrial function (Fig. 5).

Formation of ROS and mitochondrial dysfunction are also two mechanistically interrelated events [85, 86]. It is well known that cellular mitochondria are the primary source of intracellular ROS formation [86]. Excessive cellular ROS could also deteriorate proper mitochondrial function [87, 88]. Hence, oxidative stress might be a cause or a consequence of mitochondrial injury [87, 88]. Thereby, CAR and HIS could decrease ROS level not only by direct scavenging of reactive species but also by regulating sperm mitochondrial function (Fig. 5).

Acute exposure to Pb might enhance membrane susceptibility to injury and trigger a malfunction in testes and sperm by altering the major components of sperm biological membranes and polyunsaturated fatty acids. In the current study, simultaneous administration of CAR and HIS to Pb-treated rats revealed an antioxidative role and also ameliorative effects on testicular histopathological alteration against detrimental effects of Pb. Hence, the antioxidative properties of CAR and HIS might play a crucial role in scavenging the ROS induced by Pb in the testes and sperm (Fig. 5).

Metal ion chelation is an interesting feature of the peptide CAR as well as the amino acid HIS [89,90,91]. Although the capability of these chemicals for chelation of different metal ions could be variable, their metal ion-chelating properties might play a role in the mechanism of protection against Pb-induced reproductive toxicity. In the current study, a high dose of Pb (20 mg/kg/day, oral, 14 consecutive days) was administered. On the other hand, CAR and HIS were administered at least 6 h after Pb which might decrease the chance of interaction between Pb and these chemicals in the gastrointestinal tract. However, the lack of serum and testicular level of Pb might serve as a limitation for the current study. Hence, evaluating serum and tissue level of Pb, as well as CAR and HIS, might help to provide more explicit idea on their mechanism of protection against Pb toxicity.

Collectively, our data indicate that the antioxidative and mitochondria-protecting properties of CAR and HIS play a primary role in the protective properties of these molecules against Pb-induced reproductive toxicity. Therefore, CAR and HIS treatment may be used in future investigations designed for the study of reproductive toxicants where oxidative stress and mitochondrial injuries are involved.